Gastrointestinal hormones are produced by discrete neuroendocrine cells which are scattered throughout the intestine. Most GI hormone-containing cells reside within the intestinal mucosa and are often oriented with their apical region open to the lumen of the intestine. Cholecystokinin (CCK) is a prototypical gastrointestinal hormone that regulates gallbladder contraction, pancreatic enzyme secretion, delays gastric emptying, and induces satiety. As is typical of most GI hormones, CCK is secreted into the blood stream after ingestion of a meal. It is generally believed that nutrients stimulate CCK release but the cellular mechanisms regulating CCK cell function are largely unknown. Recently the PI has developed a method for isolating and characterizing individual, viable, native intestinal CCK cells and by highly enriching these cells it has been possible to study CCK secretion in vitro, identify receptors on these cells and investigate second messenger signaling pathways involved in regulated hormone secretion. Together these approaches have the ability to provide unique insights into the mechanisms by which nutrients may stimulate CCK secretion. Importantly, the PI has preliminary data that CCK cells express the calcium-sensing receptor (CaSR) and that CaSR mediates amino acid-induced CCK secretion. The PI will use complementary techniques to study the regulation of hormone secretion. These include: (1) isolation and identification of native CCK cells, (2) measurements of CCK secretion in vivo and in vitro, (3) quantification of intracellular calcium fluorescence, and (4) characterization of electrophysiological properties measured by whole-cell patch clamp recordings. The central hypothesis of this application is that gastrointestinal hormone secreting cels are electrically excitable cells whose secretion is regulated by receptor and ion channel activation. The overall purpose of this proposal is to understand the physiological regulators of GI endocrine cells with the initial focus on how amino acids control CCK secretion. Characterization of CaSR and its relationship to ion channel activation on CCK cells will be addressed by the following Specific Aims: 1. To characterize the role of CaSR in the regulation of CCK secretion in isolated CCK cells in vitro and in mice in vivo. 2. To determine effects of CaSR activation on calcium signaling in CCK cells. 3. To characterize the electrophysiological properties of CCK cells and evaluate CaSR regulation of membrane potential, and potassium channel and calcium channel activities. Each of these aims will focus on regulation of CaSR as a critical step in the regulation of amino acid-stimulated CCK secretion. More globally, these aims should provide considerable insight into the mechanisms by which GI endocrine cells are regulated by nutrients known to be important in the control of hormone secretion. PUBLIC HEALTH RELEVANCE: Cholecystokinin (CCK) is a major gastrointestinal hormone that is important in many digestive processes. Upon ingestion of a meal, CCK is released from the upper small intestine and acts on the pancreas to secrete digestive enzymes, the gallbladder to release bile, the stomach to prevent excess delivery of food to the intestine, and the brain through the vagus nerve to reduce food intake. Together these actions coordinate the ingestion and digestion of food. Despite these important effects, little is known about the control of CCK secretion. Specifically, the cellular mechanisms by which nutrients regulate CCK secretion are largely unknown. With new technologies that are now available to the PI, the current proposal will define and characterize a receptor that explains how amino acids stimulate hormone secretion. This receptor is coupled to regulation of ion channels that are involved in regulating CCK secretion. These studies should greatly expand our understanding of the nutrient regulation of hormone secretion. It is conceivable that these studies could lead to novel (luminally directed) therapies to regulate CCK release and, thereby, distant effects of CCK throughout the body (e.g., gallbladder contraction, satiety, etc.).